Hitrap q ff column

The cells were centrifuged at 4 °C and 10,000 rpm for 10 min, and the fermentation supernatant was filtered using 0.22 μm filters. ChSase AC II was purified using a HiTrap QFF column (GE Healthcare, USA). The purity of the fractions was assessed using SDS-PAGE, and the protein concentration was determined using Coomassie Brilliant Blue staining.

We optimize the candidate protein gene sequences for expression in E. coli with an added poly-his tag and TEV protease site at the N-termini and clone the genes into the pET14B vector for expression in the E. coli strain BL21 (DE3) pLysS. We grow the cells in both standard LB (Smu. 1393c and phosphoribosyl isomerase) and super broth (antigen 85-A). For the case of the catalytically active Smu. 1393c, we grow the cells in standard LB with 0.5 mM ZnCl₂. In all cases, we induce expression with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25 °C overnight for phosphoribosyl isomerase and Smu. 1393c, and 72 hours at 18 °C for antigen 85-A. The collected cells are stored at −80 °C until use.
To purify we defrost the cell pellets on ice and resuspend in buffer A (50 mM phosphate pH 7.4, 500 mM NaCl, 40 mM imidazole, 5 mM βME, and 0.1 μM Pepstatin A). We use sonication to lyse the cells and centrifuge them in a Beckman J-20 rotor at 15,000 g for 2 hours. We collect and filter the supernatant through a 0.22 μm filter before loading on a 5 mL HisTrap FF column from GE. We elute the protein with a gradient to buffer B (50 mM phosphate pH 7.4, 150 mM NaCl, 1 M imidazole). We then dialyze the proteins against 20 mM Tris pH 7.4 and elute from a HiTrap Q FF column from GE. We perform all column runs using a BioRad BioLogic low-pressure chromatography machine.

PsOep23 inclusion bodies were solubilized in 50 mM Tris–HCl pH 8.0, 100 mM NaCl, 8 M urea for 1 h at room temperature. Insoluble material was removed by centrifugation at 20,000 g for 15 min and supernatant containing PsOep23 (equiv. to 150 μg protein) was further purified using a 5 ml HisTrap HP column (GE Healthcare, Germany). Bound PsOep23 was washed with five column volumes (CV) of 50 mM Tris–HCl pH 8.0, 100 mM NaCl, 8 M urea buffer containing 35 mM imidazole. Elution was performed by raising the imidazole concentration to 500 mM.
Fractions enriched in PsOep23 were diluted 20-fold in 100 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and further purified using Strep-Tactin affinity matrix. In brief, the protein was allowed to bind to Strep-Tactin Sepharose (iba, Germany) overnight at 4°C followed by washing with 100 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% CHAPS. Protein elution was achieved by 2.5 mM desthiobiotin (iba) at 4°C for 30 min.
In a second approach nickel affinity purified PsOep23 was subjected to buffer exchange in 20 mM sodium phosphate pH 7.0, 8 M urea using Amicon centrifugal filters (MWCO 10 kDa, Merck). The PsOep23 sample was loaded onto a HiTrap Q FF column (GE Healthcare) and flow-through was collected.